The genome sequence of the Lulworth Skipper, Thymelicus acteon (Rottemburg, 1775)

We present a genome assembly from an individual male Thymelicus acteon (the Lulworth Skipper; Arthropoda; Insecta; Lepidoptera; Hesperiidae). The genome sequence is 537.0 megabases in span. Most of the assembly is scaffolded into 28 chromosomal pseudomolecules, including the Z sex chromosome. The mitochondrial genome has also been assembled and is 17.08 kilobases in length. Gene annotation of this assembly on Ensembl identified 12,813 protein-coding genes.


Background
The Lulworth Skipper (Thymelicus acteon) is a habitat specialist of warm grasslands in North Africa, Southern and Central Europe, the Middle East and Western Asia.Thymelicus acteon reaches its Northern range limit in the South of England where it occurs in a number of localities on south-facing limestone and chalk grasslands in Dorset, centred around the village of Lulworth where it was first recorded in the UK in 1832.It is absent from Wales, Scotland and Ireland.
The Lulworth Skipper is listed as Near Threatened on the IUCN and UK Red lists (Fox et al., 2015;Van Swaay et al., 2010), but in the UK it has increased in abundance since 2014, possibly as a result of climate change.The species is univoltine with adults occurring from the end of April to August depending on locality.Eggs are laid in a row in flower-sheaths of Tor-grass (Brachypodium pinnatum), the principal larval host plant in the UK, although it feeds on several other grasses across its range.
The Lulworth skipper has lower overall genetic diversity compared to its congeners T. sylvestris and T. lineola (Mackintosh et al., 2019).At the local scale in Central Europe it shows no isolation by distance, but relatively high genetic structure (Louy et al., 2007).Population connectivity has been shown to be strongly influenced by land use patterns (Engler et al., 2014).In the Western Palearctic, this species shows several geographically-structured mitochondrial lineages (Dapporto et al., 2022) as well as phenotypic variation: darker forms occur in north-west Africa, Iberia, Elba, Crete, and other eastern Mediterranean islands.The taxon christi, previously considered a subspecies of T. acteon, is now generally treated as a species endemic to the Canary Islands.

Genome sequence report
The genome was sequenced from a male Thymelicus acteon (Figure 1) collected from El Serrat,Catalunya,Spain (41.69,2.25).A total of 42-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 14 missing joins or mis-joins and removed 6 haplotypic duplications, reducing the assembly length by 0.77% and the scaffold number by 9.46%.
The final assembly has a total length of 537.0 Mb in 66 sequence scaffolds with a scaffold N50 of 19.7 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (98.98%) of the assembly sequence was assigned to 28 chromosomal-level scaffolds, representing 27 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
Two male Thymelicus acteon specimens were collected from El Serrat, Catalunya, Spain (latitude 41.69, longitude 2.25) on 2017-05-28 using a sweep net.The specimen used for genome sequencing had ID SAN00002425 (ToLID ilThyActe1), while the specimen used for Hi-C sequencing had HMW DNA was extracted in the WSI Scientific Operations core using the Automated MagAttract v2 protocol (Oatley et al., 2023).The DNA was sheared into an average fragment size of

Sequencing
Pacific Biosciences HiFi circular consensus DNA sequencing libraries were constructed according to the manufacturers' instructions.DNA sequencing was performed by the Scientific Operations core at the WSI on a Pacific Biosciences Sequel IIe instrument.Hi-C data were also generated

Software tool Version
from head tissue of ilThyActe2 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).The assembly was then scaffolded with Hi-C data (Rao et al., 2014) using YaHS (Zhou et al., 2023).The assembly was checked for contamination and corrected as described previously (Howe et al., 2021).Manual curation was performed using HiGlass (Kerpedjiev et al., 2018) and PretextView (Harry, 2022).The mitochondrial genome was assembled using MitoHiFi (Uliano-Silva et al., 2023), which runs MitoFinder (Allio et al., 2020) or MITOS (Bernt et al., 2013) and uses these annotations to select the final mitochondrial contig and to ensure the general quality of the sequence.
Table 3 contains a list of relevant software tool versions and sources.

Genome annotation
The Ensembl Genebuild annotation system (Aken et al., 2016) was used to generate annotation for the Thymelicus acteon assembly (GCA_951805285.1) in Ensembl Rapid Release at the EBI.Annotation was created primarily through alignment of transcriptomic data to the genome, with gap filling via protein-to-genome alignments of a select set of proteins from UniProt (UniProt Consortium, 2019).

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material The research only involves genomic sequencin, assembly, and annotation.These techniques are well-established in modern biological research.The genomic data obtained from the nominate subspecies will provide crucial insights into the taxonomic status of the species and its various subspecies within the genus.This study merits indexing if it meets the journal's criteria.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Systematics of Hesperiidae I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Li-Wei Wu
Tunghai University, Taichung City, Taiwan This manuscript provides a genome information about the Lulworth skipper, Thymelicus acteon.
The length of this male genome is 537 Mb in length, and total 28 chromosome-level pseudomolecules are assembled, including Z sex chromosome.The assembly quality, with an N50 of 19.7 Mb and a BUSCO completeness of 98.4%, indicates a highly complete genomic dataset.
The use of Pacific Biosciences HiFi long reads and Hi-C technology for assembly correction greatly enhances the utility of this genome for further studies in conservation genetics, evolution and phylogeography, making it an essential reference for future genomic analyses.
The analysis presented in this manuscript provides excellent genomic information.However, there are aspects of the background that could be enhanced to increase application of this manuscript and help readers, who interest this genome, understand the biology of the species more comprehensively.Two suggestions as follow: The second paragraph of the background section states, "but in the UK it has increased in abundance since 2014, possibly as a result of climate change."This claim should be supported by additional citations to clarify why the authors consider climate change a possible factor affecting the abundance of this skipper. 1.
Regarding the mitochondrial genome, which has been assembled to a length of 17-Kb, it would be beneficial to provide the GenBank accession number.This would offer convenient public access to the data, allowing others to utilize this information directly (not just only the SRA datasets).Overall, these enhancements could solidify its usefulness and accessibility.

2.
Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes

Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Entomology, conservation, evolutionary biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 1 .
Figure 1.Photographs of forewings and hindwings of the Thymelicus acteon specimen ES_TS_344 (ilThyActe1) used for genome sequencing.a) Dorsal and b) ventral surface views of wings from the specimen.

Figure 2 .
Figure 2. Genome assembly of Thymelicus acteon, ilThyActe1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 536,997,220 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (40,667,834 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (19,676,428 and 13,448,605 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilThyActe1_1/dataset/ilThyActe1_1/snail.

Figure 3 .
Figure 3. Genome assembly of Thymelicus acteon, ilThyActe1.1:BlobToolKit GC-coverage plot.Sequences are coloured by phylum.Circles are sized in proportion to sequence length.Histograms show the distribution of sequence length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilThyActe1_1/dataset/ilThyActe1_1/blob.

Figure 4 .
Figure 4. Genome assembly of Thymelicus acteon, ilThyActe1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all sequences.Coloured lines show cumulative lengths of sequences assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/ilThyActe1_1/dataset/ilThyActe1_1/cumulative.

Figure 5 .
Figure 5. Genome assembly of Thymelicus acteon, ilThyActe1.1:Hi-C contact map of the ilThyActe1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=DwelZModSOCAr3bDC4wEig.

Table 3 . Software tools: versions and sources.
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